Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq

Gilad Fuchs, Yoav Voichek, Michal Rabani, Sima Benjamin, Ido Amit, Moshe Oren

نتاج البحث: نشر في مجلةمقالةمراجعة النظراء

ملخص

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes similar to 4 d to complete, with deep sequencing requiring an additional similar to 4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.
اللغة الأصليةالإنجليزيّة
الصفحات (من إلى)605-618
عدد الصفحات14
دوريةNATURE PROTOCOLS
مستوى الصوت10
رقم الإصدار4
المعرِّفات الرقمية للأشياء
حالة النشرنُشِر - 28 أبريل 2015

All Science Journal Classification (ASJC) codes

  • !!General Biochemistry, Genetics and Molecular Biology

بصمة

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